Box-Behnken Design Approach for Optimization of a Liquid Chromatographic Method for the Determination of Anti Leukemic Drugs in Bulk and Pharmaceutical Formulations

Abstract

To develop and validate a RP-HPLC method for the determination of selected anti- leukemic drugs in bulk and pharmaceutical formulations using Design of experiments. Development of the chromatographic method is based on design of experiments (DOE) approach, utilizing two level full factorial design for screening and Box-Behnken experimental design for optimization. In order to identify the significant parameters for the optimization, by simultaneously registering the main, interaction and quadratic effects of three factors such as volume of methanol (X1), concentration of buffer(X2) and flow rate (X3) on the selected responses like capacity factor K1 of first eluted peak  (Y1), resolution of critical peaks RS (1,2) (Y2) and amp; RS (2,3) (Y3) and retention time of last peak tR4 (Y4) as responses. Analytes were separated on a Onyx monolithic- C18 (100×4.6mm) with mobile phase comprising Potassium dihydrogen orthophosphate (0.01M), Methanol and Acetonitrile in ratio of (30:34.29:35.71), with flow rate of 0.723 mL/min and pH 3.5 adjusted with dilute orthophosphoric acid. Total chromatographic analysis time per samples was approximately 5 minutes with DST(IS), IMT, IBT and SFN eluting with retention times of 2.64, 3.05, 3.81 and 4.59 minutes respectively. Calibration curves were linear over selected range 0.997 for the all analytes. The method was sensitive with the LODs were 10.457, 13.07 and 26.169 ng/mL and LOQs were 31.68, 39.6 and 79.3ng/mL for IMT, IBT and SFN respectively. Inter and Intra-day precision data (in terms of %RSD) was fond to be less than ≥3 respectively, Recoveries ranged ≥102±2% for Imatinib- Gleevec, Ibrutinib- Imbruvica and Sorafenib- Nexavar. The obtained results corroborated the potential of the proposed method for determination of all the three anti-leukemic drugs for routine analysis for products of similar type and composition.